In the field of imaging science, nothing is more challenging than imaging the world of the very small. Since the first microscopes, scientists have been fascinated with this instrument and, from its use, new discoveries and solutions to both new and old problems are made practically every day. Overall, it can be argued that microscopes have had the greatest influence in the areas of biology and medicine.
Microscope designs have stayed fairly constant over time. If one were to go into a laboratory today and dissect a modern optical microscope into its basic parts, he would find the same items that have existed in microscopes since their initial creation.
A typical optical microscope consists of a light source, a high power optical objective, a microscope body, and an eyepiece.
Nowadays the eyepiece is often replaced with a focusing lens and an electronic camera that contains an area sensor. In the history of microscopy, this fact has been a major advancement, where the human eye and sketchpad have been replaced with electronic cameras and computer aided imaging analysis.
Additional advancements have also taken place in the illuminator as well. Lasers, in conjunction with fluorescence markers, have significantly improved biologic microscopic imaging. Some of the most advanced systems involve some form of fluorescent spectroscopic imaging where laser energy is used as a narrow-band optical pump. A laser scanning confocal microscope is one such device.
For a microscope to perform well, high magnification, high contrast, and good resolution are needed. To attain this, the microscopic objective needs to have a large numerical-aperture (NA). This fact contributes to a basic limitation in most optical microscopes because having a large NA is done at the expense of reducing the operational focus range or depth-of-focus (DOF).
Visualizing a lens as an interferometer can help put this into perspective. Essentially, large NA optical systems capture highly diffracted object-generated photons with a lot of spatial information. Diffraction is fundamentally a quantum uncertainty process where the more a photon becomes localized by an object particle the more its position becomes uncertain. Hence, capturing and understanding more of a photon's diffracted information better defines the object that caused the photon to diffract in the first place. Additionally, the more a photon is localized by a particle, and consequently diffracted, the less coherent it becomes with its neighbor photons that are not so highly localized or diffracted.
Given that contrast is a function of coherence, in large NA systems out-of-focus image features will tend to blur because over a given optical statistical average, there is a higher collected ratio of incoherent to coherent photons. In essence, as you step more out-of-focus the statistical average is increasingly overwhelmed by incoherent photons, which leads to a fall off in contrast. Put another way, as NA becomes larger, contrast and associated image quality degrade more with focus error. This condition is in comparison to an optical system with the same magnification but with a smaller NA.
In short, high magnification large NA microscopes tend to have a very small DOF. Given that the microbiologic world exists and functions in a 3 dimensional space, this optical reality can be a significant challenge to any scientist. As a result, much has been invested recently, in both effort and money, to alleviate this issue.
Part of the problem is fundamental to what a geometrical optical system does with broadband light (i.e., white light). Actually, the fact that glass optics work as well as they do is a gift from nature. The fact that you can combine a few glass types, with simple spherical surface geometries, and create a quality imaging system that instantaneously and statistically integrates trillions upon trillions of remotely diffracted photons of many different energies, and doing so in-phase to within a small fraction of a wavelength, is truly an amazing thing. For intensity based imaging (which most imaging is), this process of statistical averaging works well. However, if you are interested in capturing the true phase of the photons that enter the imaging system, this method is totally unsatisfactorily.
Phase is a key property of light. Knowing the relative phase distribution (spatially and temporally) of coherently diffracted photons provides us with direct knowledge of an object's existence in four-dimensional space (three spatial dimensions and one time dimension).
Currently, one of the best commercial microscopic systems is a high-speed laser scanning, near-IR (NIR) 2-photon absorption confocal microscope, with a fast z-scanning (DOF scanning) platform or objective. This system uses 2-photon absorption fluorescent imaging to reduce background noise. These systems are very expensive, costing more than $100,000 per unit. Other microscope technologies (research grade) are exploring the use of ultra-wideband-light and near-field imaging to greatly enhance resolution. More sophisticated models can sell for up to $1,000,000 per unit.
Of these systems, none preserve the phase information of the light that is used in the imaging process. Traditionally, one needs sufficient time to measure the phase information, which means using long coherence illumination sources like a highly stabilized mode-locked laser. Here, the laser is used for direct illumination, unlike fluorescent imaging where a laser excites a secondary incoherent light source. With a long coherence illumination source, interferometric and holographic imaging are possible. However, such illumination will often result in poor image quality. Lasers, though great at enabling the determination of the phase information, can produce very poor image quality because of specular noise (typically referred to as speckle).
As is known to those skilled in the art, speckle is a phenomenon in which the scattering of light from a highly coherent source (such as a laser) by a rough surface or inhomogeneous medium generates a random intensity distribution of light that gives the surface or medium a granular appearance. Reference may be had, e.g., to page 1989 of the McGraw-Hill Dictionary of Scientific and Technical Terms, Sixth Edition (McGraw-Hill Book Company, New York, N.Y., 2003). Reference also may be had, e.g., to U.S. Pat. No. 6,587,194, the entire disclosure of which is hereby incorporated by reference into this specification.
As disclosed in U.S. Pat. No. 6,587,194: “A comprehensive description of speckle phenomena can be found in T. S. McKechnie, Speckle Reduction, in Topics in Applied Physics, Laser Speckle and Related Phenomena, 123 (J. C. Dainty ed., 2d ed., 1984) (hereinafter McKechnie). As discussed in the McKechnie article, speckle reduction may be achieved through reduction in the temporal coherence or the spatial coherence of the laser light. There have been various attempts over the years to reduce or eliminate speckle. Another article, citing the above-mentioned McKechnie article and addressing the same issues, B. Dingel et al., Speckle reduction with virtual incoherent laser illumination using a modified fiber array, Optik 94, at 132 (1993) (hereinafter Dingel), mentions several known methods for reducing speckle based on a time integration basis, as well as based on statistical ensemble integration.”
By way of further illustration, the speckle phenomenon is described at page 356 of Joseph W. Goodman's “Statistical Optics” (John Wiley & Sons, New York, N.Y., 1985), wherein it is disclosed that: “Methods for suppressing the effects of speckle in coherent imaging have been studied, but no general solution that eliminates speckle while maintaining perfect coherence and preserving image detail down to the diffraction limit of the imaging system has been found.” The present invention provides such a “general solution.”
The amount of speckle in an image may be measured in accordance with the equation set forth at page 355 of the aforementioned Goodman text (see equation 7.5-14). Reference also may be had, e.g., to U.S. Pat. No. 5,763,789 which discloses and claims: “A method for enlargement of a range of measurement of speckle measuring systems for measurement of elongation of a sample in a testing machine, comprising the steps of: providing a speckle sensor and a loading device; loading the sample into the loading device in the testing machine; and moving the speckle sensor in correspondence with a movement of the sample on the loading thereof such that a center of the field of measurement of the sample is at all times at the same object point on the loading and displacement of the sample.” The entire disclosure of this United States patent is hereby incorporated by reference into this specification.
The phase of the digital image may be calculated in accordance with the equation:
                    ζ        _            i        ⁡          (                        v          U                ,                  v          V                    )        =                    (                              I            _                    i                )            2        ⁡          [                        δ          ⁡                      (                                          v                U                            ,                              v                V                                      )                          +                                            (                                                λ                  _                                ·                                  z                  2                                            )                        2                    ⁢                                    ∫                              -                ∞                            ∞                        ⁢                          ∫                                                                                                                                      P                        ^                                            ⁡                                              (                                                  x                          ,                          y                                                )                                                                                                  2                                ⁢                                                                                                                        P                        ^                                            ⁡                                              (                                                                              x                            -                                                                                                                            λ                                  _                                                                ·                                                                  z                                  2                                                                                            ⁢                                                              v                                U                                                                                                              ,                                                      y                            -                                                                                                                            λ                                  _                                                                ·                                                                  z                                  2                                                                                            ⁢                                                              v                                V                                                                                                                                    )                                                                                                  2                                ⁢                                                      ⅆ                    x                                    ·                                      ⅆ                    y                                                                                          ]      wherein the various variables are defined in equation 7.5-14 of page 354 of Joseph W. Goodman's “Statistical Optics,” supra.
This phase, and devices or systems for measuring it, are well known. Reference may be had, e.g., to U.S. Pat. Nos. 5,541,608, 5,225,668, 4,012,689, 5,037,202, 5,789,829, 6,630,833, 3,764,897, and the like. The entire disclosure of each of these United States patent applications is hereby incorporated by reference into this specification.
As is well known to those skilled in the art, there are many companies who perform analytical services that may be utilized in making some or all of the measurements described in this specification. Thus, for example Wavefront Sciences Company of 14810 Central Avenue, S.E., Albuqurque, N. Mex. provides services including “simultaneous measurement of intensity and phase.”
Alternatively, or additionally analytical devices that are commercially available such as, e.g., the “New View 200” interferometer available from the Zygo corporation of Middlefield, Conn.
Speckle exists in incoherent imaging as well, but over the statistical block of time that an image is formed, specular artifacts are completely averaged away. This happens very quickly, on the order of femto-seconds. However, with statistical elimination of speckle, phase information is lost as well.
What is needed is time to measure the point-to-point imaged optical phase, before it's destroyed, while in the process, providing sufficient statistical information, whereby speckle is no longer an issue.
U.S. Pat. No. 5,361,131 of Tekemori et al. discloses and claims:“1. An optical displacement measuring apparatus for optically measuring a displacement amount of an object, comprising: image forming means for forming at least a first image indicative of a position of an object at a first time instant and a second image indicative of a position of the object at a second time instant; first modulating means for receiving at least the first and second images and for modulating coherent light in accordance with the first and second images, a relative position between the first image and the second image representing a displacement amount of the object achieved between the first time instant and the second time instant; first Fourier transform means for subjecting the coherent light modulated by said first modulating means to Fourier transformation to thereby form a first Fourier image; second modulating means for receiving the first Fourier image and for modulating coherent light in accordance with the first Fourier image; second Fourier transform means for subjecting the coherent light modulated by said second modulating means to Fourier transformation to thereby form a second Fourier image; detecting means for detecting a position of the second Fourier image which is indicative of the displacement amount of the object attained between the first and second time instants, said detecting means including a position sensitive light detector for receiving the second Fourier image and for directly detecting the position of the second Fourier image; and time interval adjusting means for adjusting a time interval defined between the first and second time instants, said time interval adjusting means adjusting the value of the time interval so as to cause the second Fourier image to be received by the position sensitive light detector.”
The device of the Tekemori et al. patent is not capable of eliminating specular noise in an image. The present invention can provide a digital image with a reduced amount of speckle.